The Impact of Estrogen Deficiency on Vascularization and Mineralization Dynamics—Insights from a Novel 3D Humanized and Vascularized Bone Organoid Model

Academic Background

Osteoporosis is a common bone disease, particularly prevalent among postmenopausal women. Osteoporosis not only manifests as a reduction in bone mass but also involves changes in the mineral composition of bone. Estrogen plays a crucial role in maintaining bone homeostasis, and its deficiency leads to an imbalance in bone remodeling, where bone resorption exceeds formation, resulting in bone loss. Although previous studies have revealed that estrogen deficiency affects osteoblast mineral deposition, osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis, existing in vitro bone models mostly overlook the role of vascular cells in the pathophysiology of osteoporosis. Vascularization is a key step in bone formation, especially during endochondral ossification, where the invasion of vascular cells promotes bone mineralization. However, estrogen deficiency impairs vascularization, thereby affecting bone mineralization.

To better understand the effects of estrogen deficiency on bone vascularization and mineralization, the authors developed a novel three-dimensional (3D) vascularized, mineralized, and humanized bone model to simulate postmenopausal estrogen deficiency and reveal the mechanisms underlying changes in vascularization and bone mineralization during this process. This study not only provides new insights into the pathological mechanisms of osteoporosis but also offers a potential experimental platform for future drug screening and precision medicine.

Source of the Paper

This research was conducted by Muhammad M. M. Bukhari, Mostafa Khabooshani, Syeda M. Naqvi, and Laoise M. McNamara from the Mechanobiology and Medical Device Research Group (MMDRG) at the University of Galway, Ireland. The paper was first published in the journal American Journal of Physiology-Cell Physiology on January 17, 2025, with the DOI: 10.1152/ajpcell.00738.2024.

Research Process

1. Model Development

The first step in the study was to develop a 3D vascularized, mineralized, and humanized bone model based on the endochondral ossification process. The research team used human bone marrow mesenchymal stem cells (hBMSCs) to differentiate and form a chondrogenic template in a 3D gelatin matrix. The chondrogenic template was cultured for 21 days in chondrogenic differentiation medium, followed by further culture for 42 days in a vascularized gelatin layer to promote vascularization and mineralization. The vascularized layer was formed by mixing human umbilical vein endothelial cells (HUVECs) and hBMSCs in a 1:1 ratio.

2. Simulation of Estrogen Deficiency

After the model was developed, the research team simulated postmenopausal estrogen deficiency. Both vascularized and non-vascularized bone models were cultured under estrogen-supplemented and estrogen-withdrawal conditions for 21 days, and samples were collected for analysis on days 0, 10, and 21.

3. Sample Analysis

Samples were analyzed using various experimental methods, including biochemical assays, histochemical staining, immunofluorescence staining, and real-time quantitative PCR (RT-PCR). Specific experiments included: - DNA Quantification: Used to confirm cell numbers and vascular development. - Alkaline Phosphatase (ALP) Assay: Used to evaluate osteoblast differentiation. - Calcium Content Measurement: Used to assess the degree of mineralization. - Immunofluorescence Staining: Used to detect vascular markers such as CD31 and endomucin. - Histochemical Staining: Including Alcian blue staining and von Kossa staining, used to detect glycosaminoglycans and mineralized nodules, respectively.

Key Results

1. Development of a Healthy Bone Model

The research team successfully developed a healthy 3D vascularized and mineralized bone model. Through the endochondral ossification process, the model induced self-organization of vasculature and promoted mineralization. Immunofluorescence staining confirmed the presence of CD31+ and endomucin+ endothelial cells, while histochemical staining showed the deposition of collagen II, X, and I in the chondrogenic template, indicating characteristics of early-stage endochondral ossification.

2. Effects of Estrogen Deficiency on Vascularization and Mineralization

Under estrogen-deficient conditions, distinct vessel-like structures (CD31+) appeared in the vascularized bone model, accompanied by a significant increase in mineral deposition. In contrast, no such phenomenon was observed in the non-vascularized bone model. Additionally, estrogen deficiency led to increased apoptosis in the vascularized bone model, suggesting that estrogen plays a protective role in maintaining osteocyte survival and mineralization balance.

3. The Role of Vascularization in Promoting Bone Mineralization

The study found that vascularization plays a critical role in promoting bone mineralization. Even under estrogen-deficient conditions, the vascularized bone model exhibited higher levels of mineralization, whereas the non-vascularized bone model did not. This indicates that vascularization may be one of the underlying mechanisms contributing to the heterogeneity of mineral distribution in osteoporotic bone.

Conclusion

This study presents the first 3D vascularized and humanized bone model, successfully simulating the effects of estrogen deficiency on bone vascularization and mineralization. The results indicate that estrogen deficiency exacerbates vessel formation and mineral deposition in vascularized bone models, providing a possible explanation for the heterogeneity of mineral distribution in osteoporotic bone. Furthermore, this model offers an important tool for future research on the pathological mechanisms of osteoporosis and drug screening.

Research Highlights

1. Novel Model Design: For the first time, the research team developed a 3D vascularized and humanized bone model based on the endochondral ossification process, successfully simulating postmenopausal estrogen deficiency.
2. Critical Role of Vascularization: The study reveals the crucial role of vascularization in bone mineralization, especially under estrogen-deficient conditions.
3. New Insights into Osteoporosis Mechanisms: This research provides a new mechanistic explanation for the heterogeneity of mineral distribution in osteoporotic bone, suggesting that vascularized bone is more prone to hypermineralization under estrogen-deficient conditions.

Research Value

This study not only provides new insights into the pathological mechanisms of osteoporosis but also offers a potential experimental platform for future drug screening and precision medicine. By simulating the complex bone microenvironment, this model can be used to study the mechanisms of various bone-related diseases and develop new therapeutic strategies.

Other Valuable Information

The research team also noted that future studies could further integrate mechanical stimulation and hormones (such as prostaglandins and parathyroid hormones) to more comprehensively simulate the pathological processes of osteoporosis. Additionally, the application of advanced technologies such as single-cell sequencing and spatial transcriptomics will help to deeply analyze the interactions between bone cells and vascular cells under estrogen deficiency.

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This academic news report provides a detailed introduction to the background, process, results, and conclusions of the study, emphasizing its scientific value and application potential. Through this research, we have gained a deeper understanding of the mechanisms of osteoporosis and provided new experimental tools for future research on bone-related diseases.